During the last two decades, various drug delivery systems have been designed by using carriers such as proteins, peptides, polysaccharides, synthetic polymers, colloidal particles (i.e., liposomes, vesicles or micelles), microemulsions, microspheres and nanoparticles. These carriers, which contain entrapped pharmaceutically useful agents, are intended to achieve controlled cell-specific or tissue-specific drug release.
Further efforts and research are being directed to develop and design novel systems of specific delivery to a target cell or tissue for the agents that cross biological barriers at relatively low rates. The purpose of this invention is to present a method that improves the transport and delivery characteristics of an agent molecule to a desired location, in the central nervous system,thus increasing its bioavailability.
The term "agent" as used herein relates to therapeutic, prophylactic and diagnostic compounds. These compounds are biologically active with beneficial effects in both animals and humans. Agents include lysosomal enzymes such as ceramidase, glucocerebrosidase, beta-galactosidase, beta-hexosaminidase A, beta-hexosaminidase A & B, galactosylceramidase, arylsulfatase A, sphingomyelinase, alpha-galactosidase B, aspartylglycosaminidase, alpha-L-fucosidase, iduronate sulfatase, alpha-L-iduronidase, glcNAc-6-sulfatase, beta-glucuronidase, their recombinant analogs and their derivatives. Also included are serum proteins namely immunoglobulins, interleukins, interferons, hormones, such as insulin, parathyroid hormone, pigmentary hormone, thyroid-stimulating hormones, tissue plasminogen activator, nerve growth factors, peptidases or proteases, nucleic acids and derivatives thereof, nucleotides, oligonucleotides, antisense oligonucleotide analogs, genes, transfected cells, biological vectors, cloning vectors and expression vectors. Neurotoxins or their non-toxic peptide fragments, diagnostic and research reagents are also included.
Complexes or conjugates of the above macromolecular agents with omega-3 fatty acid molecules and their derivatives are synthesized to improve transport properties, rates of distribution throughout the brain and targeted delivery of the agent to certain specific sites.
In order to exert desired therapeutic or prophylactic effects, agents must reach brain cells and tissue. Intravenous administration will require their passage from the blood to the brain by crossing the microcapillary membranes of the cerebrovascular endothelium also called the blood-brain barrier or BBB.
Briefly, the blood-brain barrier (BBB) [Johansson, Progress in Brain Research, 91: 171-175 (1992); Ermisch, Progress in Brain Research, 91: 155-161 (1992); Schlosshauer, BioEssays, 15(5): 341-346 (1993)] is formed by a monolayer of tightly connected microvascular endothelial cells with anionic charges. This layer separates two fluid-containing compartments: the blood plasma (BP) and extracellular fluid (ECF) of the brain parenchyma, and is surrounded by astroglial cells of the brain. One of the main functions of the BBB is to regulate the transfer of components between the BP and the ECF. The BBB limits free passage of most agent molecules from the blood to the brain cells.
In general, large molecules of high polarity, such as peptides, proteins, (e.g. enzymes, growth factors and their conjugates, oligonucleotides, genetic vectors and others) do not cross the BBB. Therefore poor agent delivery to the CNS limits the applicability of such macromolecules for the treatment of neurodegenerative disorders and neurological diseases.
Several delivery approaches of therapeutic agents to the brain circumvent the BBB. Such approaches utilize intrathecal injections, surgical implants [Ommaya, Cancer Drug Delivery, 1(2): 169-178 (1984) and U.S. Pat. No. 5,222,982,] and interstitial infusion [Bobo et al., Proc. Natl. Acad. Sci. U.S.A., 91: 2076-2080 (1994)]. These strategies deliver an agent to the CNS by direct administration into the cerebrospinal fluid (CSF) or into the brain parenchyma (ECF).
Drug delivery to the central nervous system through the cerebrospinal fluid is achieved by means of a subdurally implantable device named after its inventor the "Ommaya reservoir". The reservoir is used mostly for localized post-operative delivery of chemotherapeutic agents in cancers. The drug is injected into the device and subsequently released into the cerebrospinal fluid surrounding the brain. It can be directed toward specific areas of exposed brain tissue which then adsorb the drug. This adsorption is limited since the drug does not travel freely. A modified device developed by Ayub Ommaya, whereby the reservoir is implanted in the abdominal cavity and the injected drug is transported by cerebrospinal fluid (taken from and returned to the spine) all the way to the ventricular space of the brain, is used for agent administration. Through omega-3 derivatization, site-specific biomolecular complexes of this invention are overcoming the limited adsorption and movement of the agent through brain tissue. Prior art is silent on agents capable of site-directed penetration of brain tissue from the cerebrospinal fluid.
Diffusion of macromolecules to various areas of the brain by convection-enhanced delivery is another method of administration circumventing the BBB. This method consists of: a) Creating a pressure gradient during interstitial infusion into white matter to generate increased flow through the brain interstitium (convection supplementing simple diffusion); b) Maintaining the pressure gradient over a lengthy period of time (24 hours to 48 hours) to allow radial penetration of the migrating compounds (such as: neurotrophic factors, antibodies, growth factors, genetic vectors, enzymes, etc.) into the gray matter; and c) Increasing drug concentrations by orders of magnitude over systemic levels. Through their direct infusion into the brain parenchyma, the site-specific biomolecular complexes of this invention are instrumental in delivering the agent to neuronal or glial cells, as needed, and be retained by these cells. Moreover, the site-specific complexes containing neuronal targeting or internalization moieties are capable of penetrating the neuronal membrane and internalizing the agent. Prior art is silent on such site-specific delivery of macromolecular agents to specific sites in the central nervous system such as cortical and hippocampal neurons.
Another strategy to improve agent delivery to the CNS is by increasing the agent absorption (adsorption and transport) through the BBB and their uptake by the cells [Broadwell, Acta Neuropathol., 79: 117-128 (1989); Pardridge et al., J. Pharmacol. Experim. Therapeutics, 255(2): 893-899 (1990); Banks et al., Progress in Brain Research, 91: 139-148 (1992); Pardridge, Fuel Homeostasis and the Nervous System, Edited by Vranic et al., Plenum Press, New York, 43-53 (1991)]. The passage of agents through the BBB to the brain can be enhanced by improving either the permeability of the agent itself or by altering the characteristics of the BBB. Thus, the passage of the agent can be facilitated by increasing its lipid solubility through chemical modification, and/or by its coupling to a cationic carrier, or still by its covalent coupling to a peptide vector capable of transporting the agent through the BBB. Peptide transport vectors are also known as BBB permeabilizer compounds [U.S. Pat. No. 5,268,164].
Therefore, another major objective of this invention is to synthesize a site-specific macromolecule with lipophilic properties. The resulting complex or conjugate, which comprises an agent, one or more lipophilic moieties and a directing moiety, is targeted to specific cells in the brain. The lipophilic moiety is either alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) or derivatives thereof (e.g., alpha-linolenoyl-aminoethanol, N-eicosapentaenoyl-aminoethanol, N-docosahexaenoyl-amino ethanol, or alpha linolenoyl-ethanolamine, eicosapentaenoyl-ethanolamine, docosahexaenoyl-ethanolamine, or lipids of all three omega-3 acids).
There is significant transport of poly-unsaturated fatty acids of the omega-3 series (PUFA, omega-3) across the BBB that is possibly mediated by a specific transporter and/or a receptor or simply by the transcytosis process. Prior art discloses that the BBB can target the release of omega-3 fatty acids into the brain and thus aid in the enrichment of these essential fatty acids in the brain [Yerram et al., J. Lipid Research, 30: 1747-1757 (1989); Moore et al., J. Neurochem., 55(2): 391-402 (1990); Moore et al. J. Neurochem., 56(2):518-524 (1991)]. Also, prior art indicates that the microvascular endothelium (the BBB) and astrocytes, and not neurons, are responsible for the elongation and desaturation of alpha-linolenic acid and eicosapentaenoic acids respectively in the rodent brain (Moore et al., ibid). Moreover, astrocytes are apparently responsible for the elongation and desaturation of EPA into docosahexaenoic acid (DHA, or C22:6, omega-3), the release of the latter into the extracellular space or transfer to neurons. Whichever the mechanism, the DHA ends up in the neuronal membrane in form of phospholipids. If the DHA is synthesised in the liver or administered intravenously, then it apparently crosses the BBB to the brain. Therefore, the DHA enrichment in the brain may result from three important roles of the BBB: (1) to take up ALA from the blood, transform it into EPA and eliminate the latter into the perivascular glial cells of the brain, (2) take up DHA from the blood and release it into the brain and (3) to block the egress of DHA from the brain. The latter is probably responsible for the relatively high DHA content of brain phosphotriglycerides when compared to other polyunsaturated fatty acids (N. Salem et al., in Health Effects of Polyunsaturated Fatty Acids in Seafoods. A. P. Simopoulos, ed. Academic Press, New York, 1986, p. 263-317 and G. Y. Sun and L. L. Foudin, in "Phospholipids in Nervous Tissue", J. E. Eichenberg, ed. John Wiley & Sons, New York 1985, p. 95-115. Therefore, EPA directs the agent to the glia and DHA directs the agent to cortical, cholinergic and adrenergic neurons. ALA will reach the microvascular endothelium where it will be transformed into EPA. The use of lipophilic moieties could further protect the agent from enzymatic degradation during its passage across the BBB.
Prior art shows, for example, conjugates of proteins with water soluble polymers which have been manufactured and used for pharmaceutical purposes. U.S. Pat. No. 4,935,465 teaches the attachment of one or more polymer molecules to a protein. However, attachment of polymers to proteins, particularly to enzymes which act on low molecular weight substrates, retards enzyme clearance and decreases enzyme antigenicity. When the enzyme acts on a macromolecular substrate or on a cell-bound substrate, the enzyme activity of the conjugate could be diminished.
Other examples [U.S. Pat. No. 4,701,521, and U.S. Pat. No. 4,847,240] describe a method of covalently bonding an agent to a cationic macromolecular carrier which enters into the cells at relatively higher rates. These patents teach enhancement in cellular uptake of bio-molecules into the cells when covalently bonded to cationic resins.
U.S. Pat. No. 4,046,722 discloses anti-cancer drugs covalently bonded to cationic polymers for the purpose of directing them to cells bearing specific antigens. The polymeric carriers have molecular weights of about 5,000 to 500,000.
Further work involving covalent bonding of an agent to a cationic polymer through an acid-sensitive intermediate (called also spacer) molecule, is described in U.S. Pat. No. 4,631,190 and U.S. Pat. No. 5,144,011. Various spacer molecules, such as cis-aconitic acid, are covalently linked to the agent and to the polymeric carrier. They control the release of the agent from the macromolecular carrier when subjected to a mild increase in acidity, such as probably occurs within a lysosome of the cell. The drug can be selectively hydrolyzed from the molecular conjugate and released in the cell in its unmodified and active form. Molecular conjugates are transported to lysosomes, where they are metabolised under the action of lysosomal enzymes at a substantially more acidic pH than other compartments or fluids within a cell or body. The pH of a lysosome is shown to be about 4.8, while during the initial stage of the conjugate digestion is possibly 3.8.
U.S. Pat. No. 5,308,701 discloses a method for encapsulating proteins within a synthetic cationic poly-L-lysine, crosslinked with multivalent ions of the opposite charge, to form a hydrolytically stable gel.
In general, both natural and synthetic polymers have been used as drug carriers. Synthetic cationic polymers are more suitable drug carriers than natural polymers because their structure can be systematically altered in a defined way and therefore, it is possible to design them to suit biological requirements such as penetration through the blood-brain barrier.
In the present invention, the polymeric cationic carrier system used to facilitate the crossing of the blood-brain barrier comprises poly-L-lysine (PLL). PLL is a bio-compatible, hydrophilic polymer with very thoroughly studied chemical and biological properties. Other cationic polyamino acids such as polyarginine and polyornithine are within the scope of this invention. These polyamino acids are covalently attached to a biopolymeric agent conjugated with omega-3 fatty acid molecules or other polymeric protecting groups (e.g. polyethylene glycol). In addition, we have such complexes/conjugates covalently attached to omega-3 molecules for improved binding by the microvasular endothelial cell membrane and targeting to specific areas of the brain. A nontoxic fragment of a neurotoxin is further attached to assure targeting and internalization by neurons once the neuronal target is reached. Spacers can be inserted between the components of the vehicle as shown in the description of the preferred embodiments.
When the biopolymeric agent is an enzyme molecule, one or more fatty acid moieties (e.g. ALA, EPA or DHA) can be attached to the enzyme by reacting an imidate activated enzyme with the respective fatty acid. The number of attached moieties is limited only by the number of available --NH.sub.2 groups of the enzyme.
The prior art is silent on lipophilic macromolecular conjugates, potentially containing polycationic amino acids (targeting lipophilic macromolecular vehicles), used to deliver therapeutic or diagnostic agents across the BBB selectively to specific areas of the brain.